Andreas Rolfs

The ABCs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteins

The Human Genome Organisation (HUGO) Nomenclature Committee Database provides a list of transporter families of the solute carrier (SLC) gene series (see http://www.gene.ucl.ac.uk/nomenclature/). Currently, it includes 43 families and 298 transporter genes. This special issue features mini-reviews on each of these SLC families written by the experts in each field. A WEB site has been established (http://www.pharmaconference.org/slctable.asp) that gives the latest updates for the SLC families and their members as well as relevant links to gene databases and reviews in the literature. A list of all currently known SLC families, a discussion of additional SLC families and family members as well as a brief summary of non-SLC transporter genes is included in this introduction.

Next-generation high-density self-assembling functional protein arrays

We developed a high-density self-assembling protein microarray, based on the nucleic acid programmable protein array (NAPPA) concept, to display thousands of proteins that are produced and captured in situ from immobilized cDNA templates. We arrayed up to 1,000 unique human cDNAs and obtained high yields of protein expression and capture with minimal variation and good reproducibility. This method will enable various experimental approaches to study protein function in high throughput.

Protein Structure Initiative Material Repository: an open shared public resource of structural genomics plasmids for the biological community

The Protein Structure Initiative Material Repository (PSI-MR; http://psimr.asu.edu) provides centralized storage and distribution for the protein expression plasmids created by PSI researchers. These plasmids are a resource that allows the research community to dissect the biological function of proteins whose structures have been identified by the PSI. The plasmid annotation, which includes the full length sequence, vector information and associated publications, is stored in a freely available, searchable database called DNASU (http://dnasu.asu.edu). Each PSI plasmid is also linked to a variety of additional resources, which facilitates cross-referencing of a particular plasmid to protein annotations and experimental data. Plasmid samples can be requested directly through the website. We have also developed a novel strategy to avoid the most common concern encountered when distributing plasmids namely, the complexity of material transfer agreement (MTA) processing and the resulting delays this causes. The Expedited Process MTA, in which we created a network of institutions that agree to the terms of transfer in advance of a material request, eliminates these delays. Our hope is that by creating a repository of expression-ready plasmids and expediting the process for receiving these plasmids, we will help accelerate the accessibility and pace of scientific discovery.

The ORFeome Collaboration: a genome-scale human ORF-clone resource

To the Editor: Here we describe the ORFeome Collaboration (OC) open reading frame (ORF) clone collection, created by the OC (http://www.orfeomecollaboration.org/), an international collaboration of academic and commercial groups committed to providing genome-scale clone resources for human genes via worldwide commercial and academic clone distributors. Proteins are the predominant functional modules determining the fate of cells, tissues and organisms. An encyclopedic understanding of cellular physiology requires protein expression for proteinprotein interaction screening, cellular functional screening, validation of knockout and knockdown phenotypes, and numerous other approaches. Performing such studies on individual proteins or at the proteome scale requires a comprehensive collection of human protein expression clones. Our collection comprises ORF clones (Supplementary Note) and covers 17,154 RefSeq and Ensembl genes, nearly 73% of human RefSeq genes (http://www.ncbi.nlm.nih.gov/refseq/rsg/) and 79% of the highly curated Consensus Coding DNA Sequence Project (CCDS) human genes (http://www.ncbi.nlm.nih.gov/CCDS/ CcdsBrowse.cgi) (Fig. 1a and Supplementary Data). The collection includes clones of transcript variants for 6,304 (37%) of those genes. All major functional categories of human genes are substantially represented (Fig. 1b). All clones are provided in the Gateway vector format (Life Technologies), permitting high-throughput, precise and directional transfer of ORFs to a large variety of vectors for protein expression in biological systems such as Escherichia coli, yeast and mammals or using cell-free protein expression1 (Supplementary Note). OC clones were generated primarily by PCR amplification of the ORF from full-length, sequence-verified human cDNA clones of the Mammalian Gene Collection2 or the German cDNA Consortium3; ORFs were also prepared by directed RT-PCR cloning4 or DNA synthesis2. All 5′ and 3′ untranslated regions were excluded, permitting direct expression of ORFs as fusions to aminoor carboxy-terminal polypeptides, or as native protein, after transfer to a Gatewayexpression vector1. The clones are designed to maintain the correct reading frame for both aminoand carboxy-fusion proteins. Among all genes represented in the OC collection, 64% of clones are without stop codons, 5% have stop codons, and 31% are present in both versions. Each OC clone was isolated from a single colony and is fully sequenced. Individual clone sequences have been deposited in the GenBank, EMBL and DDBJ databases. The OC website provides a searchable database with annotation of all OC clones, their respective genes, and clone confidence levels based on CCDS and RefSeq annotations (Supplementary Note) along with links to the UCSC and RIKEN browsers (http://genome.ucsc.edu/cgi-bin/hgGateway and http://fantom.gsc.riken.jp/zenbu/gLyphs/#config), which provide graphical representations of the gene structures and transcripts. OC clones are distributed via a good faith agreement, giving unrestricted clone access to all scientists worldwide. The OC website lists OC clone distributors. The value of the OC resource has been demonstrated in numerous studies covering a broad range of applications. These include large-scale binary protein-protein interaction mapping5, production of recombinant human proteins6, mapping of co-complex associations, fluorescent protein tagging for human protein localization in mammalian cells and microscopy-based functional screening of proteins, development of disease-specific protein interaction Figure 1 | RefSeq and Ensembl genes and functional gene categories represented in the OC. (a) Numbers of protein-coding genes represented in the OC collection from RefSeq (blue) and Ensembl (green) gene catalogs. The table summarizes these numbers, together with OC coverage for RefSeq-only and Ensembl-only genes. (b) Numbers of human RefSeq genes represented in the OC collection versus in the human genome, compared in nine functional categories; percentages of genes in the OC are presented above the bars. The methods used to calculate the gene numbers in each category are explained in the Supplementary Note and contrasted to the standard Gene Ontology categories. An expanded list of the top Gene Ontology categories is also provided in the Supplementary Note. The data underlying the graphs are provided as Supplementary Data. a

Production and sequence validation of a complete full length ORF collection for the pathogenic bacterium Vibrio cholerae

Cholera, an infectious disease with global impact, is caused by pathogenic strains of the bacterium Vibrio cholerae. High-throughput functional proteomics technologies now offer the opportunity to investigate all aspects of the proteome, which has led to an increased demand for comprehensive protein expression clone resources. Genome-scale reagents for cholera would encourage comprehensive analyses of immune responses and systems-wide functional studies that could lead to improved vaccine and therapeutic strategies. Here, we report the production of the FLEXGene clone set for V. cholerae O1 biovar eltor str. N16961: a complete-genome collection of ORF clones. This collection includes 3,761 sequence-verified clones from 3,887 targeted ORFs (97%). The ORFs were captured in a recombinational cloning vector to facilitate high-throughput transfer of ORF inserts into suitable expression vectors. To demonstrate its application, ≈15% of the collection was transferred into the relevant expression vector and used to produce a protein microarray by transcribing, translating, and capturing the proteins in situ on the array surface with 92% success. In a second application, a method to screen for protein triggers of Toll-like receptors (TLRs) was developed. We tested in vitro-synthesized proteins for their ability to stimulate TLR5 in A549 cells. This approach appropriately identified FlaC, and previously uncharacterized TLR5 agonist activities. These data suggest that the genome-scale, fully sequenced ORF collection reported here will be useful for high-throughput functional proteomic assays, immune response studies, structure biology, and other applications.

PlasmID: a centralized repository for plasmid clone information and distribution

The Plasmid Information Database (PlasmID; ) was developed as a community-based resource portal to facilitate search and request of plasmid clones shared with the Dana-Farber/Harvard Cancer Center (DF/HCC) DNA Resource Core. PlasmID serves as a central data repository and enables researchers to search the collection online using common gene names and identifiers, keywords, vector features, author names and PubMed IDs. As of October 2006, the repository contains >46 000 plasmids in 98 different vectors, including cloned cDNA and genomic fragments from 26 different species. Moreover, the clones include plasmid vectors useful for routine and cutting-edge techniques; functionally related sets of human cDNA clones; and genome-scale gene collections for Saccharomyces cerevisiae, Pseudomonas aeruginosa, Yersinia pestis, Francisella tularensis, Bacillus anthracis and Vibrio cholerae. Information about the plasmids has been fully annotated in adherence with a high-quality standard, and clone samples are stored as glycerol stocks in a state-of-the-art automated −80°C freezer storage system. Clone replication and distribution is highly automated to minimize human error. Infor-mation about vectors and plasmid clones, including downloadable maps and sequence data, is freely available online. Researchers interested in requesting clone samples or sharing their own plasmids with the repository can visit the PlasmID website for more information.

A Full-Genomic Sequence-Verified Protein-Coding Gene Collection for Francisella tularensis

The rapid development of new technologies for the high throughput (HT) study of proteins has increased the demand for comprehensive plasmid clone resources that support protein expression. These clones must be full-length, sequence-verified and in a flexible format. The generation of these resources requires automated pipelines supported by software management systems. Although the availability of clone resources is growing, current collections are either not complete or not fully sequence-verified. We report an automated pipeline, supported by several software applications that enabled the construction of the first comprehensive sequence-verified plasmid clone resource for more than 96% of protein coding sequences of the genome of F. tularensis, a highly virulent human pathogen and the causative agent of tularemia. This clone resource was applied to a HT protein purification pipeline successfully producing recombinant proteins for 72% of the genes. These methods and resources represent significant technological steps towards exploiting the genomic information of F. tularensis in discovery applications.

A Biomedically Enriched Collection of 7000 Human ORF Clones

We report the production and availability of over 7000 fully sequence verified plasmid ORF clones representing over 3400 unique human genes. These ORF clones were derived using the human MGC collection as template and were produced in two formats: with and without stop codons. Thus, this collection supports the production of either native protein or proteins with fusion tags added to either or both ends. The template clones used to generate this collection were enriched in three ways. First, gene redundancy was removed. Second, clones were selected to represent the best available GenBank reference sequence. Finally, a literature-based software tool was used to evaluate the list of target genes to ensure that it broadly reflected biomedical research interests. The target gene list was compared with 4000 human diseases and over 8500 biological and chemical MeSH classes in ∼15 Million publications recorded in PubMed at the time of analysis. The outcome of this analysis revealed that relative to the genome and the MGC collection, this collection is enriched for the presence of genes with published associations with a wide range of diseases and biomedical terms without displaying a particular bias towards any single disease or concept. Thus, this collection is likely to be a powerful resource for researchers who wish to study protein function in a set of genes with documented biomedical significance.